1. Field of the Invention
The present invention relates to techniques for producing ions for the determination of molecular weights of chemical compounds by mass spectrometry. More particularly, the invention relates to improved electrospray ionization techniques for producing molecular ions at atmospheric pressure, and for efficiently transmitting such ions into the vacuum of a mass spectrometer without introducing uncontrolled changes in mass due to fragmentation or clustering with solvent or other neutral molecules.
2. Description of the Background
Mass spectrometry is a well known technique for obtaining a molecular weight and structural information on chemical compounds. According to mass spectrometry, molecules may be "weighed" by ionizing the molecules and measuring the response of their trajectories in a vacuum to electric and magnetic fields. With traditional ionization techniques such as electron ionization, photo-ionization, and chemical ionization, the applications of mass spectrometry were limited to relatively low molecular weight, thermally stable neutral molecules.
Improved "soft" ionization techniques, such as field desorption, thermospray, and electrospray, have recently been developed which produce intact molecular ions from high molecular weight, ionic and thermally labile molecules. As a result, applications of mass spectrometry have become increasingly important in many areas, such as biological research, where detection and characterization of such materials is often required. While each of these soft ionization techniques may be preferred for certain applications, electrospray is one of the most promising techniques for producing molecular weight information on large biopolymers, such as proteins.
Electrospray ionization techniques were first proposed in the late 1960's. A sample solution containing molecules of interest and a solvent is pumped through a hypodermic needle and into an electrospray chamber. An electrical potential of several kilovolts may be applied to the needle for generating a fine spray of charged droplets. According to UK patent specification 1,246,709, the droplets may be sprayed at atmospheric pressure into a chamber containing a heated gas to vaporize the solvent. Alternatively, the needle may extend into an evacuated chamber, and the sprayed droplets then heated in the evacuated chamber by an infared filament. In either case, ions are focused into a beam, which is accelerated by an electric field gradient, and the ions then analyzed in a mass spectrometer.
Significant disadvantages are encountered if an electrospray is discharged into an evacuated chamber. The charged droplets are not retarded from migrating toward the chamber walls, thereby increasing the possibility of discharge and disruptions to the spray. U.S. Pat. No. 4,209,696 teaches an electrospray technique which occurs at atmospheric pressure or above, and the produced ions are input to a mass analyzer.
Although the electrospray is preferably formed at atmospheric pressure, mass spectrometers operate within a vacuum chamber. A vacuum housing for a mass spectrometer typically includes a plurality of lenses in a vacuum chamber, which is maintained at a sufficiently low pressure that collisions of the ions with neutral molecules is unlikely. The chamber is typically heated to about 100.degree. C. to prevent condensation on the lenses and to keep them clean. When a gas at atmospheric pressure passes through a small orifice into an evacuated chamber, it expands rapidly in all directions and the pressure in the expanding gas decreases in proportion to the square of the distance from the orifice inside the vacuum chamber. The role of the lenses is to focus the ions into a beam for analysis by the mass analyzer, but the lenses are relatively ineffective at influencing the trajectory of the ions until the pressure in the expanding gas is sufficiently low that the mean free path for collisions of ions with neutral molecules is larger than the critical dimensions of the lenses. As a result, a large fraction of the ions produced in the electrospray chamber may be swept away by the expanding gas and removed by the vacuum pumps. Only a small fraction of the produced ions are focused by the lenses and transmitted to the mass analyzer for detection. Accordingly, this low transfer of ions to the mass analyzer produced by electrospray substantially limits the sensitivity of the electrospray/mass spectrometer technique.
Another significant problem with electrospray concerns the condensation of the expanding jet and clustering of the ions. To reduce this problem, heated counterflow gases are commonly employed to vaporize sprayed droplets and desolvate ions at atmospheric pressure. Since the heated counterflow gases remove much of the solvent vapor from the stream of gas before being drawn into the vacuum chamber, this technique increases the concentration of ions of interest in the vacuum chamber. U.S. Pat. No. 4,023,398 teaches a technique whereby ions pass through an orifice into a vacuum chamber, while a gas curtain upstream from the orifice reduces transmission of solvent vapor into the vacuum chamber. The gas is heated to hasten evaporation of the solvent from the droplets, thereby producing desolvated ions at substantially atmospheric pressure U.S. Pat. No. 4,531,056 teaches a similar technique, whereby an inert gas is introduced into the electrospray chamber in a direction opposite to a flow from the capillary. The electrospray chamber remains at or slightly greater than atmospheric pressure. Ions of interest are produced within the electrospray chamber, and the inert gas flow substantially reduces the concentration of solvent vapor which enters the analyzer. U.S. Pat. Nos. 4,842,701 and 4,885,076 disclose a system which combines capillary zone electrophoresis with electrospray for gas analysis of an analyte mixture. Again, the electrospray occurs at atmospheric pressure, and a heated countercurrent gas flow technique is used to desolvate the spray droplets.
While the counterflowing gas concept described above results in reasonable sensitivity, it substantially increases the complexity of the interface between the electrospray and the mass spectrometer. In order that the solvent vapor from the evaporating droplets be efficiently removed by the counterflowing gas, both the temperature and the flow rate of the gas must be carefully controlled. High gas flow rates may prevent some ions with low mobility from entering the analyzer, while low gas flow rates or reduced gas temperature may not sufficiently desolvate the ions. The counterflowing gas flow rate and temperature are typically optimized for each analyte and solvent. Accordingly, much trial and error time is necessary to determine the optimum gas flow rate and temperature for each particular analyte utilizing a particular electrospray device and a particular mass spectrometer.
A major limitation of these prior art electrospray devices is that, for a given set of operating conditions, a stable electrospray can only be achieved within a rather narrow range of liquid input flow rates and liquid compositions. In particular, these prior art techniques are not practical for electrospraying pure water, or water containing modest amounts of ionic buffers. As a result, these systems cannot be used effectively with standard liquid chromatography procedures commonly used in gradient elution from reversed phase, which require continuous modification of the mobile phase composition from 100% water to either 100% methanol or acetonitrile.
The disadvantages of the prior art are overcome by the present invention, and improved techniques are hereinafter disclosed for improving the stability, reliability, and sensitivity of electrospray mass spectrometry so that it can be readily coupled to standard micro-bore HPLC techniques for the analysis of a wide variety of biological and other materials.